Two Coccidian Parasites and An Unusual Intraerythrocytic Cytoplasmic Vacuolar Inclusion from Agamid Lizard, Trapelus savignii in Egypt

Three types of parasites, two intraerythrocytic and one Isospora species, were recorded to infect lizard Trapelus savignii (Reptilia: Agamidae) collected in summer 2019 from the Eastern Desert in Egypt. The current intraerythrocytic parasites are haemogregarine species and unusual intraerythrocytic cytoplasmic vacuolar inclusions (ICVIs). Blood stages of haemogregarine species are differentiated into three forms; trophozoite, intermediate form, and mature gamont. Trophozoites measure 7.50–8.50 (8.10±0.30) × 2.20–2.40 (2.30±0.06) μm while intermediate forms and mature gamonts measure 7.89–10.39 (9.32±0.85) × 3.9–5.18 (4.32±0.45) and 8.87–12.13 (10.25±0.90) × 2.50–4.35 (3.45± 0.58) μm,respectively. ICVIs are un- or pale-stained with Giemsa and measure 1.62–5.45 (3.64±0.87) × 1.45–5.35 (3.51±1.10) μm. At the ultrastructure level, ICVIs appeared as a membrane-bound electron-lucent matrix and contain three types of materials according to their electron density that may resemble chromatin granules. No nucleus, Golgi apparatus, mitochondria, or any apical complex structure is observed within ICVIs. Effects of haemogregarine species and ICVIs on the infected erythrocytes are recorded. Exogenous and endogenous developmental stages of the present Isospora sp. are described. Sporulated oocysts are mostly spherical, contain bilayered wall, and measure 22.48–27.71 (25.47±1.81) × 21.69–26.91 (24.37±1.73). Oocyst residuum, polar granule and micropyle are absent. Sporocysts are ellipsoidal and measure 13.79–15.84 (14.97±0.67) × 8.80–11.03 (9.97±0.70). Stieda body is nipple-like while sub-Stieda body is absent. Multinucleate meronts contain up to 34 nuclei and measure 6.29–19.83 (11.91±4.58) × 5.43–10.76 (7.57±1.75). Developed microgamonts measure 16.59–18.89 (17.74±0.94) × 15.61–18.63 (17.12±1.24) while mature macrogamonts measure 16.34–24.27 (19.95±2.98) × 12.0–20.28 (16.92±3.22).


INTRODUCTION
Haemogregarines represent a group of heteroxenous intracellular blood parasites and are classified in suborder Adeleorina, order Eucoccidiorida, class Coccidia within the apicomplexan alveolates (Votýpka et al., 2017). These protists include eight genera and their vertebral (intermediate) hosts are recorded from fishes to mammals (Davies and Johnston, 2000;Telford, 2009).
Based on biological data and phylogenetic reconstruction, Karadjian et al. (2015) proposed a new classification for the terrestrial haemogregarines that extends to include Hepatozoon infecting mammalian carnivores, Karyolysus infecting reptiles, Hemolivia infecting reptiles and amphibians, in addition to the creation of a new genus, Bartazoon, infecting amphibians, reptiles, birds, and rodents. However, Maia et al. (2016) suggested that is still premature to finalize the systematic revision of haemogregarines as more molecular and biological data are needed. In Egypt, many haemogregarines were reported from lacertilian and ophidian hosts under the generic name Haemogregarina "sensu lato" (s.l.) and Hepatozoon (Bashtar et al., 1984(Bashtar et al., , 1987Abdel-Ghaffar et al., 1994;Al-Hoot and Abd-Al-Aal, 1999;Abdel-Haleem et al., 2013).
The taxonomic status of intraerythrocytic cytoplasmic vacuolar inclusions (ICVIs) of lizards is a matter of controversy and their presence apparently shows low biodiversity among lacertilian reptiles. The majority of intraerythrocytic viral and bacterial inclusions are densely redstained in Giemsa-stained blood films while so-called piroplasm inclusions are un-or pale-stained (Telford, 2009). Du Toit (1937 proposed a new piroplasmid genus, Sauroplasma to include small, rounded (typically ring-shaped) or irregularly shaped unpigmented intracytoplasmic parasites in erythrocytes of lizards. Telford (2009) defined reptilian piroplasmids as "chromatin granules associated with a vacuole or resemble a ring" and stated that Sauroplasma and Serpentoplasma, if they indeed piroplasms, show a little affinity to the mammalian piroplasms. Some authors assumed that the presence of so-called Sauroplasma spp. in lizards was overlooked or mistaken as artifacts or viral or bacterial infections and vice versa (Davies and Johnston, 2000;Telford, 2009;Ursula et al., 2014).
Regarding the number of species, Isospora Schneider, 1881 is considered as the second largest biodiverse genus within the family Eimeriidae, after Eimeria Schneider, 1875 (Ghimire, 2010). Isospora species belong to order Eucoccidiorida, class Coccidia, phylum Apicomplexa, and most of their descriptions are based mainly on sporulated oocysts morphology (Berto et al., 2011;Votýpka et al., 2017). The sporulated oocysts of reptilian Isospora spp. are characterized by having two sporocysts with Stieda bodies and each sporocyst contains four sporozoites (Barta et al., 2005) (Bashtar et al., 1984(Bashtar et al., , 1987Shazly, 2000; Mansour and Abou Shafeey, 2019) but not from agamid lizard Trapelus savignii yet. However, a suggested new Caryospora sp. is reported from lizard Trapelus savignii, recently (Mansour, 2020). The current study aims to investigate and discuss the characteristic features of intraerythrocytic blood parasites and developmental stages of Isospora species from Savigny's Agama Trapelus savignii in Egypt.

MATERIALS AND METHODS Experimental Animals:
One hundred and three agamid lizards Trapelus savignii Duméril and Bibron, 1837 (Agamidae: Sauria: Reptilia) were collected in summer 2019 from the Egyptian Eastern Desert and transported to the laboratory of Parasitology and invertebrates at Zoology and Entomology Department, Faculty of Science, Helwan University, where they were investigated for the presence of blood parasites and coccidian infections.

Light microscopic studies:
Morphological characterizations of the current intraerythrocytic parasites and Isospora species were examined and photographed under an oil immersion lens (1000X) using a Zeiss research photomicroscope.
Measurements are expressed in micrometers (μm) and presented by the range of length × width and then followed by the mean values ± standard deviation in parentheses.

1-Examination of blood parasites:
Thick and thin blood films were prepared from lizard tails. Blood smears were air-dried, fixed in absolute methanol (5 min), stained with 3% phosphate-buffered Giemsa stain (pH 7.3) for 30 minutes, dehydrated in ascending series of ethanol, cleared in xylene, and mounted in Canada balsam.

2-Examination of isosporan infection: a-Investigation of oocysts (exogenous stages):
The abdomen of each lizard was gently squeezed to get fecal particles. Obtained faeces were carried by forceps on a clean slide. The investigation of the present isosporan oocysts was carried out by direct fecal smears or by mixing and diluting of faeces in a drop of distilled water. The oocysts in intestinal contents of Isosporapositive lizards are concentrated by flotation technique following Long et al. (1976).

b-Investigation of merogonic and gamogonic stages (endogenous stages):
Pieces measuring about 5 mm were taken from the stomach, small intestine, liver, kidney, spleen and rectum of the Isospora-infected lizards and were immediately fixed in 10% phosphatebuffered formalin (pH 7.3) for at least 12 hours at 4 0 C. After 3-4 washings in the phosphate buffer for 10-15 min each, the specimens were dehydrated in ethanol ascending series, cleared in xylene, embedded in parablast and sectioned by a rotatory microtome at thickness of 5 µm. Then, sections were deparaffinized, hydrated in ethanol descending series, stained with haematoxylin and eosin stain, dehydrated in ascending ethanol series and cleared in xylene again, and finally covered by a coverslip with Canada balsam.

Transmission electron microscopy (TEM) of ICVIs:
Blood samples of the infected lizards were primary fixed in 3% (v/v) glutaraldehyde buffered in 0.1M sodium cacodylate buffer (pH 7.3) for at least 4 hours. Then, fixed specimens were washed in buffer three times (15 min for each) and secondary fixed with 1% osmium tetroxide buffered in 0.1M sodium cacodylate buffer for 2 hours. Afterward, specimens were dehydrated in ascending ethanol series, embedded in epoxy resin, sectioned into semithin sections (1 µm thick), and stained with toluidine blue stain. Ultra-thin sections (50-80 nm thick) were cut using a Leica Ultracut UCT Ultramicrotome, stained with uranyl acetate for 20 min and alkaline lead citrate 2-3 min (Reynolds 1963) and finally examined by a JEOL-JEM 1010 TEM at the

Reda M. Mansour
```` 4 Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.

RESULTS I-Intraerythrocytic parasites (Figs. 1 and 2): A-Haemogregarine species (Figs. 1a-f): (1)-Prevalence and level of parasitaemia:
Out of one hundred and three examined lizards Trapelus savignii, only one male (0.97 %) was found to be naturally infected with haemogregarine sp. on the initial examination. The level of parasitaemia (numbers of haemogregarine-infected erythrocytes/1000 erythrocytes) was generally low and recorded as 1.80 %.
(2)-Blood stages of the parasite: The blood stages of the present haemogregarine are differentiated into three forms; the small, intermediate, and mature forms. The nucleus of parasite is usually obvious and the cytoplasm is free of granules or vacuoles. The small form or trophozoite (n = 12) is the thinnest stage and has a nucleus located near its pointed end while the opposite end is broad (Fig. 1a) Parasitophorous vacuoles are scarcely noticed in most cases between the parasite and host cell cytoplasm.
No extracellular or leukocytic stages are noticed. Normally, the Two Coccidian Parasites and An Unusual Intraerythrocytic Cytoplasmic Vacuolar Inclusion from Agamid Lizard, 5 infected blood cell contains only one ICVI (Figs. 1d, g and h), but occasionally, up to four ICVIs are seen within the cytoplasm of a single erythrocyte (Fig. 1h). Under light microscopy, these ICVIs are un-or palestained with Giemsa and vary in shape from crescent rod-shape to triangular, quadrate, spherical, subspherical, oval and irregular shape. ICVIs (n = 100) measure 1.62-5.45 (3.64±0.87) × 1.45-5.35 (3.51±1.10) and in some cases, they are seen in erythrocytes infected by the present haemogregarine (Fig.  1d).
(3)-Transmission electron microscopy (TEM) of ICVIs ( Fig. 2): At ultrastructural levels, ICVIs acquired many different shapes. They are nearly ovoid with a shape index of 1.12-1.23 (Figs. 2a-c, g and i), ellipsoid with shape index of 1.41-1.58 (Figs. 2d-f), slipper-like (Figs. 2h and j), kidney-shaped (Fig. 2k) or resemble bean seed embryo (Fig. 2l). Some ICVIs are seen neighboring eleven relatively small vacuoles (Fig. 2a). However, many ICVIs are noticed to have envelopes thickened by moderate electron-dense materials (Figs. 2b,c,e,f,i,k,and l). Within the electron-lucent matrix of these ICVIs, three types of materials are noticed according to their electron density. They are highly, moderately, or low electron-dense materials that may resemble chromatin granules.
The highly electron-dense materials are found in form of relatively fine dispersed particles (Figs. 2a, c, g, h, k and l), rounded globules (Fig. 2e), or short beadedlike string (Fig. 2c). The highly electrondense materials are mostly seen associated with moderately electron-dense materials (Figs. 2a, c, e, f and l). The envelope of one ICVI is seemed to invaginate and internalize some of the exposed substances in behavior that resembles endocytosis (Fig. 2b). However, another ICVI is seemed to be in endo-or exo-cytic process (Fig. 2i). Low electron-dense materials are seen in some ICVIs (Figs. 2d and e). In addition, some ICVIs are observed in a process resembling budding or fission (Fig. 2g) while others have process-like structures (Figs. 2k and i).

(4)-Effects of ICVIs on infected erythrocytes:
Under light microscopy, most of the ICVIs-contained erythrocytes are of normal size and shape but some are slightly wider than normal (Figs. 1g and h)
Developed microgamonts are noticed and characterized by the presence of many randomly distributed small nuclei (Figs. 3n and o). Microgamonts with 12-16 nuclei (Fig. 3n)  Developed and mature macrogamonts are also described (Figs. 3p and q). Developed macrogamont is characterized by the beginning of the appearance of wall forming bodies (Fig. 3p). It has a subspherical shape, relatively small size, central nucleus, and measures 6.02-9.55 (7.79±1.44) × 5.51-8.97 (7.24±1.41) with a shape index of 1.06-1.09 (10.8±0.01). Mature macrogamont is characterized by having a larger number of wall bodies that may reach up to 60 (Fig. 3q) Egypt (Bashtar et al., 1987;Abdel-Ghaffar et al., 1994;Al-Hoot and Abd-Al-Aal, 1999;Abdel-Haleem et al., 2013) and the present species is recorded for the first time from the agamid lizard Trapelus savignii in Egypt. It represents one of the terrestrial haemogregarines but in absence of molecular data, it is hard to locate it under a specific generic name. The prevalence of present haemogregarine sp. is low for a reptilian host recording 0.97 %. The present parasite is compared with other haemogregarines reported from some saurian hosts in prevalence, parasitaemia level, measurements of intraerythrocytic forms, and effects on infected erythrocytes (Table  1). Three erythrocytic blood forms are  Al-Aal, 1999;Paperna et al., 2002). However, only two blood forms are also reported (Bashtar et al., 1987;Abdel-Ghaffar et al., 1994;Shazly, 2000) (see Table 1). Like the majority of terrestrial haemogregarines (Table 1), mature gamonts of the present species are typically bananashaped with non-pigmented cytoplasmic granules. In addition, single erythrocytic infection is only recorded and intraerythrocytic merogony is absent. Similar observations were reported in many reptiles infected by Haemogregarina and Hepatozoon species (Al-Hoot and Abd-Al- Aal, 1999;Shazly, 2000;Abdel-Haleem et al., 2013;Van As et al., 2013). However, leucocytes were recorded as the main site of gamont infection for mammalian haemogregarines (Smith, 1996). In addition, Hussein (2006) recorded single and double haemogregarine infections in both erythrocytes and leucocytes of the infected gecko Ptyodactylus hasselquistii from Qena in Egypt. Double infection was also noticed for some haemogregarine infections (Bashtar et al., 1987;Al-Hoot and Abd-Al-Aal, 1999;Abdel-Haleem et al., 2013;Abou Shafeey et al., 2019).

Many haemogregarines are recorded from lacertilian hosts in
The present species causes slight erythrocyte hypertrophy and erythrocytic nucleus elongation that displaced laterally or terminally.
ICVIs of the present lizard are rarely-or un-stained. Some authors follow the suggestion of Du Toit (1937) and attribute pale-or un-stained ICVIs of lizards to genus Sauroplasma (Svahn, 1976;Alberts et al., 1998;Telford, 2009;Ursula et al., 2104). Ultrastructure findings of the present ICVIs showed the absence of nucleus, mitochondria, Golgi apparatus and other structures distinctive for eukaryotic cell and this may support the hypothesis proposed by Davies and Johnston (2000) that genus Sauroplasma is not of protistan origin. However, all examined transmission electron micrographs of the current study showed no packed viral or bacterial particles within these ICVIs. In addition, the presence of a process resembling endo-or exo-cytosis for the present ICVIs is considered a matter of controversy as this behavior is characteristic for eukaryotic cells and scarcely found in bacteria (Fuerst and Sagulenko, 2010;Mills, 2020). Moreover, three types of materials according to their electron density that may resemble chromatin granules were seen dispersed in the electron-lucent matrix of the present ICVIs. Furthermore, electron micrographs do not reveal any apicomplexan structures. Similar observations were noticed in transmission electron micrographs for other so-called Sauroplasma spp. (Alberts et al., 1998;Van As, 2012).
The endogenous stages of current Isospora sp. occur in the cytoplasm of enterocytes and lamina propria of the small intestine of the infected lizards taking an apical position to enterocytes nucleus. Similar results were reported in I. ameivae (Lainson and Paperna, 1999), I. pardali (Abdel-Ghaffar et al., 2015) and I. vittati (Mansour and Abou Shafeey, 2019). However, intranuclear development was recorded for I. cannoni, I. choochotei, I. deserti (Finkelman and Paperna, 1994) and I. hemidactyli (Lainson and Paperna, 1999). The present meronts contained up to 34 nuclei and were seen containing up to 10 merozoites. Different shapes and measurements of meronts with different number nuclei and merozoites were observed for many Isospora species (Finkelman and Paperna, 1994;Lainson and Paperna, 1999;Abdel-Ghaffar et al., 2015;Mansour and Abou Shafeey, 2019